Use of anoctamin as a biomarker for radiation biodosimetry

ABSTRACT

In one embodiment, the present invention pertains to the use of anoctamin as a biomarker for determining radiation dosimetery. In certain embodiments, the present invention relates to the use of anoctamin as a biomarker for diagnosing the presence of radiation toxicity in a subject who has been exposed to ionizing radiation, as well as for determining the absorbed radiation dose in a subject who has been exposed to a known or unknown dose of ionizing radiation. In another embodiment, the expression level of anoctamin can be used as a secondary endpoint to determine mechanisms of action and/or pharmacodynamic (PD) effects of an agent for reducing radiation toxicity.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser.No. 61/710,413, filed Oct. 5, 2012, which is incorporated herein byreference in its entirety.

The Sequence Listing for this application is labeled“SeqList-07Oct13_ST25.txt”, which was created on Oct. 7, 2013, and is 15KB. The entire content is incorporated herein by reference in itsentirety.

BACKGROUND OF INVENTION

Radiation therapy, a common treatment regime for cancer, can causesevere damage to radiosensitive organs, including the bone marrow, thegastrointestinal (GI) tract, and the lung. Toxic effects of radiation onthe gastrointestinal system cause symptoms such as nausea, vomiting,diarrhea, electrolyte imbalance, and dehydration. Radiation can alsocause pulmonary injury, leading to pulmonary pneumonitis and fibrosis.

Radiation toxicity not only causes devastating effects on the quality ofpatient life, but can sometimes even be more life-threatening than theprimary tumor or cancer. Therefore, it is important to monitor theseverity of radiation toxicity in patients during the course ofradiation therapy.

Currently, there is no biomarker that indicates whether a persondevelops radiation toxicity. There is also no biomarker that canaccurately determine the radiation dose absorbed by a person havingradiation exposure.

The anoctamin (ANO, also known as TMEM16) protein family, which consistsof 10 members (ANO 1-10) in mammals, is a family of transmembraneproteins having Ca²⁺-activated Cl⁻ activity. ANO proteins play a role invarious diseases including cancer. It has been reported that ANO1 (alsoknown as TMEM16a) is upregulated in gastrointestinal stromal tumor, aswell as in oral and head and neck squamous cell carcinoma. It has beenalso reported that ANO5 (also known as TMEM16e) mutations in humanscause gnathodiaphyseal dysplasia. In addition, it has been reported thatANO7 (also known as TMEM16g) is selectively expressed in normal andcancerous prostates and regulates cell-cell aggregation.

Anoctamin proteins have not previously been reported as being associatedwith radiation toxicity.

BRIEF SUMMARY

In one embodiment, the present invention pertains to the use ofanoctamin as an early biomarker for radiation biodosimetery. In aspecific embodiment, the present invention relates to the use ofanoctamin as a biomarker for diagnosing the presence of radiationtoxicity in a subject who has been exposed to ionizing radiation.

In another embodiment, the present invention relates to the use ofanoctamin as a biomarker for determining the absorbed radiation dose ina subject who has been exposed to a known or unknown dose of ionizingradiation.

Advantageously, the diagnostic and prognostic assays of the presentinvention are rapid, sensitive, and non-invasive. The present inventioncan be useful in civilian and military industries.

In one embodiment, the present invention provides a method fordetermining radiation dose absorbed by a subject who has been, or issuspected of having been, exposed to ionizing radiation, wherein themethod comprises:

(a) providing a biological sample from a subject who has been, or issuspected of having been, exposed to ionizing radiation;

(b) determining anoctamin expression level in the subject's biologicalsample; and

(c) determining the radiation dose absorbed by the subject based on thelevel of expression determined in step (b).

In another embodiment, the present invention provides a method ofdetermining whether a subject has radiation toxicity, wherein the methodcomprises:

(a) providing a biological sample from a subject who has been, or issuspected of having been, exposed to ionizing radiation;

(b) determining anoctamin expression level in the subject's biologicalsample; and

(c) comparing the expression level determined in step (b) to a level ofanoctamin expression in a normal control;

wherein an increased expression of anoctamin in the subject's biologicalsample with respect to the control indicates that the subject hasradiation toxicity.

In one embodiment, the absorbed radiation dose and/or the presence ofradiation toxicity is determined based on the ANO1 expression level in abiological sample. In specific embodiments, the biological sample is ablood sample (such as whole blood, plasma, and serum).

In another embodiment, expression level of anoctamin can be used as asecondary endpoint to determine mechanisms of action and/orpharmacodynamic (PD) effects of an agent for reducing radiationtoxicity.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 (A-C) shows the anoctamin 1 protein in normal, non-irradiatedmouse cells.

FIG. 2 (A-F) shows the anoctamin 1 protein in mouse cells 6 days afterthe mice received irradiation at 3 Gy, 5 Gy, and 7 Gy, respectively.

FIG. 3 shows anoctamin 1 protein expression level in mouse red bloodcells 6 days after the mice received irradiation at 3 Gy, 5 Gy, and 7Gy, respectively.

FIG. 4 shows changes in Isc with time since 3-Gy irradiation. Thehighest current was seen at 94 hours.

FIG. 5 shows changes in Isc with increasing dose of irradiation. Asignificant increase in Isc was seen in 3, 5, and 7 Gy compared to 0 Gyirradiated mice (P<0.01). All results are from n=6 tissues. Error barsrepresent standard error of mean (SEM).

FIG. 6 shows the effect of irradiation dose on ANO1 protein levels.Ileal tissues were collected on day 6 following irradiation. Irradiationresulted in a dose-dependent increase in ANO1 protein levels.

FIG. 7 shows the effect of irradiation dose on ANO1 protein level in redblood cell (RBC) ghost membrane on day 6 post-irradiation.

FIG. 8 shows the effect of irradiation on intracellular cAMP andintracellular calcium in mouse ileal epithelial cells. Radiationincreases intracellular calcium and not cAMP. All measurements were madeon day 6 following 3 Gy irradiation.

FIG. 9 shows immunohistochemistry sections for ANO1 expression in mousesmall intestine. (A) and (B) show ANO1 expression in mouse ileum from 0Gy irradiated mice. Most of the expression occurred in the villousepithelium with minimal expression in the brush border membrane region.(C) and (D) show ANO1 expression in mouse ileum from 3 Gy irradiatedmice. ANO1 expression shows higher intensity in the brush bordermembrane region of villous cells. (E) and (F) show ANO1 expression inmouse ileum from 5 Gy irradiated mice. 5 Gy irradiated mice showed ahigher degree of expression for the ANO1 chloride channel than tissuesfrom 0 Gy and 3 Gy irradiated mice. The ANO1 protein was mostlyexpressed in the villous cell region with minimal expression in thecrypt cell region.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO:1 is the amino acid sequence of a human anoctamin 1 protein(GenBank Accession No. NP_(—)060513).

SEQ ID NO:2 is the nucleic acid sequence of a human anoctamin 1 mNRAtranscript (GenBank Accession No. NM_(—)018043).

DETAILED DISCLOSURE

In one embodiment, the present invention relates to the use of anoctaminas a biomarker for diagnosing the presence of radiation toxicity,specifically, radiation-induced acute gastrointestinal toxicity, in asubject who has been exposed to radiation (such as ionizing radiation).In another embodiment, the present invention relates to the use ofanoctamin as a biomarker for determining the absorbed radiation dose ina subject who has been exposed to a known or unknown dose of radiation(such as ionizing radiation).

In another embodiment, the present invention relates to the use ofanoctamin as a biomarker for determining effectiveness of a therapy forreducing radiation toxicity. In one embodiment, the expression level ofanoctamin can be used as a secondary endpoint to determine mechanisms ofaction and/or pharmacodynamic (PD) effects of an agent for reducingradiation toxicity.

After irradiation, glucose transport is partially or completelydown-regulated in a dose-dependent manner. As a result, oral glucoseintake after irradiation would activate calcium-activated electrogenicchloride secretion, thereby resulting in secretory diarrhea. Westernblot analysis of the small intestinal mucosa of mice exposed toirradiation shows increased anoctamin-1 expression even on day 6post-irradiation. Anoctamin-1 expression level is also increased in themembrane of red blood cells (RBCs) after irradiation.

In one embodiment, the present invention provides a method fordetermining radiation dose absorbed by a subject who has been, or issuspected of having been, exposed to ionizing radiation, wherein themethod comprises:

(a) providing a biological sample from a subject who has been, or issuspected of having been, exposed to radiation (such as ionizingradiation);

(b) determining expression level of an anoctamin (such as anoctamin 1)in the subject's biological sample; and

-   -   (c) determining the radiation dose absorbed by the subject based        on the level of expression determined in step (b).

In another embodiment, the present invention provides a method ofdetermining whether or not a subject has radiation toxicity, wherein themethod comprises:

(a) providing a biological sample from a subject who has been, or issuspected of having been, exposed to radiation (such as ionizingradiation);

(b) determining expression level of an anoctamin (such as anoctamin 1)in the subject's biological sample; and

-   -   (c) comparing the expression level determined in step (b) to a        level of an anoctamin (such as anoctamin 1) expression in a        normal control;

wherein an increased expression of an anoctamin (such as anoctamin 1) inthe subject's biological sample with respect to the control indicatesthat the subject has radiation toxicity.

In one embodiment, an increased expression of an anoctamin (such asanoctamin 1) in the subject's biological sample with respect to thecontrol indicates that the subject has radiation-induced acutegastrointestinal toxicity.

In a further embodiment, the present invention provides a method ofdetermining whether a subject has developed radiation toxicity duringthe course of radiation therapy, wherein the method comprises:

(a) providing a biological sample from a subject who has been prescribedradiation therapy at a predetermined dose;

(b) before radiation therapy, determining expression level of ananoctamin (such as anoctamin 1) in a biological sample of the subject;

(c) providing radiation therapy to the subject at the predetermineddose;

(d) determining expression level of an anoctamin (such as anoctamin 1)in the subject's biological sample after the subject has been exposed toradiation at the predetermined dose;

(e) comparing the expression level determined in step (d) to theanoctamin (such as anoctamin 1) expression level determined in step (b);and

(d) if the level of anoctamin (such as anoctamin 1) expressiondetermined in (d) is at least 105%, 110%, 115%, 120%, 125%, 130%, 135%,140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, or 500% ofthe anoctamin (such as anoctamin 1) expression level determined in step(b), then the subject has radiation toxicity.

In certain embodiments, the present invention can be used to determinethe absorbed radiation dose and/or determine the presence of radiationtoxicity 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after thesubject has received, or is suspected of receiving, irradiation.

In certain embodiments, anoctamin (e.g., ANO 1) expression level isdetermined using a biological sample obtained no more than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the subject hasreceived, or is suspected of receiving, ionizing radiation.

In certain embodiments, anoctamin (e.g., ANO 1) expression level isdetermined using a biological sample obtained no more than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the subject hasreceived, or is suspected of receiving, radiation at a dose of at least0.5 Gy or higher (including, but not limited to, at least 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, and90 Gy).

In one embodiment, the absorbed radiation dose and/or the presence ofradiation toxicity is determined based on the ANO1 expression level in abiological sample. In one embodiment, the biological sample is a bloodsample (such as whole blood, plasma, and serum). In one embodiment, thelevel of an anoctamin (e.g., ANO 1) in the membranes of RBCs of asubject is determined.

In a further embodiment, the anoctamin expression level in a subject isdetermined at multiple time points to determine whether the subject hasradiation toxicity, to monitor the severity of radiation toxicity,and/or to determine the treatment effects of a therapeutic regime forreducing radiation toxicity.

The term “subject,” as used herein, describes an organism, includingmammals such as primates. Mammalian species that can benefit from thedisclosed methods of treatment include, but are not limited to, apes,chimpanzees, orangutans, humans, monkeys; and other animals such asdogs, cats, horses, cattle, pigs, sheep, goats, chickens, mice, rats,guinea pigs, and hamsters. In one embodiment, the subject is a human.

In one embodiment, the subject has been exposed to radiation during thecourse of radiation therapy for tumor or cancer. In another embodiment,the subject has been, or is suspected of having been, exposed toionizing radiation by accident.

In certain embodiments, the subject has been exposed to radiation (suchas via prescription during ionizing radiation therapy) or is suspectedof having been exposed to radiation (such as by accidental exposure toionizing radiation) at a dose of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, or 90 Gy.In certain embodiments, the subject has been exposed to radiation (suchas via prescription during ionizing radiation therapy) or is suspectedof having been exposed to radiation (such as by accidental exposure toionizing radiation) at a dose of at least 0.1, 0.3, 0.5, 0.7, 0.9, 1.0,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4,2.5, 2.7, 3.0, 3.2, 3.5, or 4.0 Gy in one day.

In a further embodiment, the present invention provides a method ofproviding a toxicity-monitored radiation therapeutic regime, wherein themethod comprises:

(a) providing a biological sample from a subject who has been exposed toradiation therapy at a predetermined dose;

(b) determining anoctamin (e.g., ANO 1) expression level in thesubject's biological sample after the subject has been exposed toradiation at the predetermined dose;

(c) comparing the expression level determined in step (b) to a level ofanoctamin (e.g., ANO 1) expression in a normal control;

(d) if the level of anoctamin (e.g., ANO 1) expression determined in (b)is greater than control, then prescribing additional radiation at a doselower than the predetermined dose, discontinuing radiation therapy forat least 1 day or any days longer than 1 day (including, but not limitedto, at least 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 30 days,60 days, 90 days, and 180 days), or prescribing a therapy that reducesradiation-induced toxicity (such as radiation-induced acutegastrointestinal toxicity); and

if the level of anoctamin (e.g., ANO 1) expression determined in (b) isno greater than the control, then continuing radiation therapy at a doseidentical to, or higher than, the predetermined dose.

In a further embodiment, the present invention provides a method ofproviding a toxicity-monitored radiation therapeutic regime, wherein themethod comprises:

(a) providing a biological sample from a subject who has been prescribedradiation therapy at a predetermined dose;

-   -   (b) before radiation therapy, determining anoctamin (e.g.,        ANO 1) expression level in a biological sample of the subject;

(c) providing radiation therapy to the subject at the predetermineddose;

(d) determining anoctamin (e.g., ANO 1) expression level in thesubject's biological sample after the subject has been exposed toradiation at the predetermined dose;

(e) comparing the expression level determined in step (d) to theanoctamin (e.g., ANO 1) expression level determined in step (b);

(f) if the level of anoctamin (e.g., ANO 1) expression determined in (d)is at least 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 150%, 160%,170%, 180%, 190%, 200%, 250%, 300%, 400%, or 500% of the anoctamin(e.g., ANO 1) expression level determined in step (b), then prescribinga second radiation dose lower than the predetermined dose, discontinuingradiation therapy for at least 1 day or any days longer than 1 day(including, but not limited to, at least 2 days, 3 days, 4 days, 5 days,10 days, 15 days, 30 days, 60 days, 90 days, and 180 days), orprescribing a therapy that reduces radiation-induced toxicity (such asradiation-induced acute gastrointestinal toxicity); and

if the level of anoctamin (e.g., ANO 1) expression determined in (d) isno greater than 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 150%,160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, or 500% of the anoctamin(e.g., ANO 1) expression level determined in step (b), then continuingthe prescribed radiation dose.

In certain embodiments, in the course of providing a toxicity-monitoredradiation therapeutic regime, anoctamin (e.g., ANO 1) expression levelis determined 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 daysafter the subject has received the predetermined radiation dose. Theanoctamin (e.g., ANO 1) expression level can be determined at multipletime points over time. In certain embodiments, therapies that reduceradiation-induced toxicity (such as radiation-induced acutegastrointestinal toxicity) include, but are not limited to, oralrehydration compositions, and compositions disclosed inPCT/US2011/053265, which is hereby incorporated by reference in itsentirety.

The term “biological sample,” as used herein, includes, but is notlimited to, a sample containing tissues, cells, and/or biological fluidsisolated from a subject. Examples of biological samples include but, arenot limited to, tissues, cells, biopsies, blood, lymph, serum, plasma,urine, saliva, and tears. In one embodiment, the biological samplecontains red blood cells.

In one embodiment, the absorbed radiation dose and/or the presence ofradiation toxicity is determined based on expression level(s) of one ormore anoctamins selected from the group consisting of anoctamin 1,anoctamin 2, anoctamin 3, anoctamin 4, anoctamin 5, anoctamin 6,anoctamin 7, anoctamin 8, anoctamin 9, and anoctamin 10.

The level of anoctamin expression can be determined based on mRNA levelsor protein levels. Determination of anoctamin expression can be madequalitatively, semi-quantitatively, or quantitatively. Sequences ofanoctamin proteins and mRNAs of a variety of mammalian species arepublicly available and can be obtained from, for example, the GenBankdatabase. In one embodiment, the human anoctamin 1 protein has the aminoacid sequence of SEQ ID NO:1 (GenBank Accession No. NP_(—)060513). Inanother embodiment, the human anoctamin 1 mNRA transcript has thenucleic acid sequence of SEQ ID NO:2 (GenBank Accession No.NM_(—)018043). One of ordinary skill in the art, having the benefit ofthe present disclosures, can easily use anoctamin protein and nucleicacid sequences of a mammalian species of interest to practice thepresent invention.

In one embodiment, the control level of anoctamin expression isdetermined by measuring anoctamin expression in a healthy populationthat has not been exposed to radiation (such as ionizing radiation)and/or does not have acute or long term side effects caused byirradiation.

Methods for determining anoctamin expression level are well known in theart, including but not limited to, Western blot, enzyme-linkedimmunosorbent assay (ELISA), immunoprecipitation, polymerase chainreaction (PCR) methods including reverse transcription polymerase chainreaction (RT-PCR), nucleic acid hybridization, and any combinationthereof. In a preferred embodiment, the anoctamin expression level isdetermined using ELISA.

A contacting step in the assay (method) of the invention can involvecontacting, combining, or mixing the biological sample and a solidsupport, such as a reaction vessel, microbeads, microvessel, tube,microtube, well, multi-well plate, or other solid support.

An antibody that specifically recognizes, or specifically binds to,anoctamin proteins (e.g., ANO1) can be in any of a variety of forms,including intact immunoglobulin molecules, fragments of immunoglobulinmolecules such as Fv, Fab and similar fragments; multimers ofimmunoglobulin molecules (e.g., diabodies, triabodies, and bi-specificand tri-specific antibodies, as are known in the art; see, e.g., Hudsonand Kortt, J. Immunol. Methods 231:177 189, 1999); fusion constructscontaining an antibody or antibody fragment; and human or humanizedimmunoglobulin molecules or fragments thereof.

“Specific binding” or “specificity” refers to the ability of a proteinto detectably bind an epitope presented on a protein or polypeptidemolecule of interest, while having relatively little detectablereactivity with other proteins or structures. Specificity can berelatively determined by binding or competitive binding assays, using,e.g., Biacore instruments. Specificity can be exhibited by, e.g., anabout 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greaterratio of affinity/avidity in binding to the specific target moleculeversus nonspecific binding to other irrelevant molecules.

Antibodies within the scope of the invention can be of any isotype,including IgG, IgA, IgE, IgD, and IgM. IgG isotype antibodies can befurther subdivided into IgG1, IgG2, IgG3, and IgG4 subtypes. IgAantibodies can be further subdivided into IgA1 and IgA2 subtypes.

Antibodies of the present invention include polyclonal and monoclonalantibodies. The term “monoclonal antibody,” as used herein, refers to anantibody or antibody fragment obtained from a substantially homogeneouspopulation of antibodies or antibody fragments (i.e. the individualantibodies within the population are identical except for possiblenaturally occurring mutations that may be present in a small subset ofthe antibody molecules).

In one embodiment, the level of anoctamin (e.g., ANO1) proteinexpression is determined by contacting the biological sample with anantibody that specifically recognizes, or specifically binds to, ananoctamin protein (e.g., ANO1); and detecting the complex formed betweenthe antibody and the anoctamin (e.g., ANO1) protein.

The level of anoctamin (e.g., ANO1) expression can be determined basedon anoctamin (e.g., ANO1) mRNA level. In one embodiment, the anoctaminmRNA level can be determined by a method comprising contacting thebiological sample with a polynucleotide probe that comprises a nucleicacid sequence that specifically binds to, or hybridizes under stringentconditions with, an anoctamin (e.g., ANO 1) mRNA; and detecting thecomplex formed between the polynucleotide probe and the anoctamin (e.g.,ANO1) mRNA.

As used herein, “stringent” conditions for hybridization refers toconditions wherein hybridization is typically carried out overnight at20-25° C. below the melting temperature (Tm) of the DNA hybrid in6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Themelting temperature, Tm, is described by the following formula (Beltz etal., 1983):

Tm=81.5C+16.6 Log [Na+]+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes are carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at Tm-20 C for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

In one embodiment, the anoctamin mRNA level can be determined bypolymerase chain reaction methods. Polymerase chain reaction (PCR) is aprocess for amplifying one or more target nucleic acid sequences presentin a nucleic acid sample using primers and agents for polymerization andthen detecting the amplified sequence. The extension product of oneprimer when hybridized to the other becomes a template for theproduction of the desired specific nucleic acid sequence, and viceversa, and the process is repeated as often as is necessary to producethe desired amount of the sequence. The skilled artisan, to detect thepresence of a desired sequence (U.S. Pat. No. 4,683,195), routinely usespolymerase chain reaction.

A specific example of PCR that is routinely performed by the skilledartisan to detect desired sequences is reverse transcript PCR (RT-PCR;Saiki et al., Science, 1985, 230:1350; Scharf et al., Science, 1986,233:1076). RT-PCR involves isolating total RNA from biological fluid,denaturing the RNA in the presence of primers that recognize the desirednucleic acid sequence, using the primers to generate a cDNA copy of theRNA by reverse transcription, amplifying the cDNA by PCR using specificprimers, and detecting the amplified cDNA by electrophoresis or othermethods known to the skilled artisan.

Samples and/or anoctamin (e.g., ANO1)-specific binding agents may bearrayed on a solid support, or multiple supports can be utilized, formultiplex detection or analysis. “Arraying” refers to the act oforganizing or arranging members of a library (e.g., an array ofdifferent samples or an array of devices that target the same targetmolecules or different target molecules), or other collection, into alogical or physical array. Thus, an “array” refers to a physical orlogical arrangement of, e.g., biological samples. A physical array canbe any “spatial format” or “physically gridded format” in which physicalmanifestations of corresponding library members are arranged in anordered manner, lending itself to combinatorial screening. For example,samples corresponding to individual or pooled members of a samplelibrary can be arranged in a series of numbered rows and columns, e.g.,on a multi-well plate. Similarly, binding agents can be plated orotherwise deposited in microtitered, e.g., 96-well, 384-well, or1536-well plates (or trays). Optionally, anoctamin (e.g., ANO1)-specificbinding agents may be immobilized on the solid support.

In another embodiment, the present invention provides a method forscreening for a therapeutic agent that reduces radiation toxicity,wherein the method comprises:

(a) providing a population of cells that have been exposed to radiation(such as ionizing radiation) and have an increased expression ofanoctamin (e.g., ANO1), and optionally, determining a first level ofanoctamin (e.g., ANO1) expression in the population of cells exposed toradiation (such as ionizing radiation);

(b) contacting the population of cells with a candidate therapeuticagent for reducing radiation toxicity;

(c) after step (b), determining anoctamin (e.g., ANO1) expression levelin the population of cells contacted with the candidate therapeuticagent; and

(d) selecting the candidate agent that reduces the level of anoctamin(e.g., ANO1) expression as the therapeutic agent that reduces radiationtoxicity.

In another embodiment, the present invention provides a method foridentifying an agent that increases radiation toxicity, wherein themethod comprises:

(a) providing a population of cells that have been exposed to radiation(such as ionizing radiation) and have an increased expression ofanoctamin (e.g., ANO1), and optionally, determining a first level ofanoctamin (e.g., ANO1) expression in the population of cells exposed toradiation (such as ionizing radiation);

(b) contacting the population of cells with a candidate agent;

(c) after step (b), determining anoctamin (e.g., ANO1) expression levelin the population of cells contacted with the candidate agent; and

(d) identifying the candidate agent that increases the level ofanoctamin (e.g., ANO1) expression, when compared to the first level ofanoctamin (e.g., ANO1) expression, as an agent that increases radiationtoxicity.

In a further embodiment of the screening method, the candidate agent iscontacted with a population of cells of a subject who has been exposedto radiation (such as ionizing radiation).

Kits

The present invention provides kits comprising the required elements fordetecting anoctamins (e.g., ANO1).

In one embodiment, the present invention provides a kit for determiningwhether the subject has radiation toxicity, for determining the absorbedradiation dose, for monitoring the severity of radiation toxicity,and/or for determining the treatment effects of a therapeutic regime forreducing radiation toxicity.

In certain specific embodiments, the kit comprises an application zonefor receiving a biological sample (such as a blood sample); a labelingzone containing a binding agent that binds to an anoctamin (e.g., ANO 1)protein or mRNA in the sample; and a detection zone where anoctamin(e.g., ANO 1)-bound binding agent is retained to give a signal, whereinthe signal given for a sample of a subject with an anoctamin (e.g.,ANO 1) level greater than a control level is different from the signalgiven for a sample of a subject with an anoctamin (e.g., ANO 1) levellower than a control level.

In one embodiment, the kit comprises an anoctamin-binding agentincluding, an antibody that specifically recognizes, or specificallybinds to, an anoctamin protein (e.g., ANO1); a polynucleotide probe thatcomprises a nucleic acid sequence that specifically binds to, orhybridizes under highly stringent condition to, an anoctamin (e.g.,ANO1) mRNA; and a primer set that amplifies an anoctamin (e.g., ANO1)mRNA.

Preferably, the kits comprise a container for collecting samples, suchas blood samples, from a subject, and an agent for detecting thepresence or the level of anoctamin (e.g., ANO 1) in the sample. Theagent may be any binding agent specific for anoctamin (e.g., ANO1),including, but not limited to, antibodies, aptamers, nucleic acidprobes, and primers. The components of the kit can be packaged either inaqueous medium or in lyophilized form.

As indicated above, kits of the invention include reagents for use inthe methods described herein, in one or more containers. The kits mayinclude specific internal controls, and/or probes, buffers, and/orexcipients, separately or in combination. Each reagent can be suppliedin a solid form or liquid buffer that is suitable for inventory storage.Kits may also include means for obtaining a sample from a host organismor an environmental sample.

Kits of the invention can be provided in suitable packaging. As usedherein, “packaging” refers to a solid matrix or material customarilyused in a system and capable of holding within fixed limits one or moreof the reagent components for use in a method of the present invention.Such materials include glass and plastic (e.g., polyethylene,polypropylene, and polycarbonate) bottles, vials, paper, plastic, andplastic-foil laminated envelopes and the like. Preferably, the solidmatrix is a structure having a surface that can be derivatized to anchoran oligonucleotide probe, primer, molecular beacon, specific internalcontrol, etc. Preferably, the solid matrix is a planar material such asthe side of a microtiter well or the side of a dipstick. In certainembodiments, the kit includes a microtiter tray with two or more wellsand with reagents including primers, probes, specific internal controls,and/or molecular beacons in the wells.

Kits of the invention may optionally include a set of instructions inprinted or electronic (e.g., magnetic or optical disk) form, relatinginformation regarding the components of the kits and/or how to makevarious determinations (e.g., anoctamin levels, comparison to controlstandards, etc.). The kit may also be commercialized as part of a largerpackage that includes instrumentation for measuring other biochemicalcomponents.

EXAMPLES

Following are examples that illustrate procedures and embodiments forpracticing the invention. The examples should not be construed aslimiting.

Example 1 Radiation Increases Anoctamin-1 Expression in Red Blood Cells

Anoctamin-1 expression level is increased in the membrane of red bloodcells (RBCs) after irradiation. Briefly, RBC ghosts are prepared frommice that have been exposed to different radiation doses, and Westernanalysis is performed.

FIG. 1 (A-C) shows the anoctamin 1 protein in normal, non-irradiatedmouse cells. FIG. 2 (A-F) shows the anoctamin 1 protein in mouse cells 6days after the mice received irradiation at 3 Gy, 5 Gy, and 7 Gy,respectively. FIG. 3 shows anoctamin 1 protein expression level in mousered blood cells 6 days after the mice received irradiation at 3 Gy, 5Gy, and 7 Gy, respectively.

As shown in FIG. 3, the Western analysis shows that there is a radiationdose-dependent increase in anoctamin-1 expression at a radiation dose ofup to about 5 Gy, while there is a marginal decrease in anoctamin-1expression at a radiation dose of about 7 Gy; it is postulated that 7 Gycauses increased cell death in the RBC cells used in the currentexperiments, thereby resulting in a marginal decrease in anoctamin-1expression. Nevertheless, the anoctamin expression at a radiation doseof 7 Gy is higher than that of the non-irradiated mice.

Example 2 ANO1 A Biomarker for Radiation-Induced Acute GastrointestinalToxicity

Total body irradiation (TBI) results in a dose-dependent increase ingastric anion secretion. This Example shows that the expression level ofANO1 in RBC indicates acute gastrointestinal radiotoxicity.

Materials and Methods Animal

Eight-week old NIH Swiss mice were used in this study.

Ussing Chamber

Murine ileal sections were used for transepithelial electrical currentmeasurements after 0, 12, 24, 48, 96, 192, and 384 hours after TBI.Tissues were mounted in Ussing chambers, bathed in modified regularRinger's solution, and gassed with 95% O₂ & 5% CO₂ to measure shortcircuit current (Isc), a measure of anion secretion. Basal readings weretaken after 45 minutes, and the peak current was measured in chambersunder different conditions (NSP4 and glucose+NSP4). The CaCC inhibitorniflumic acid was used to study the role of chloride secretion throughCaCC.

RBC Ghosts

RBC ghosts were used, and changes in anoctamin 1 (ANO1) expression inRBC ghosts were determined.

Immunohistochemistry

Mice were irradiated with 0 Gy, 3 Gy, or 5 Gy, and ileal tissuescollected on day 6 following irradiation. Harvested samples wereimmediately fixed in buffered formalin, embedded in paraffin, andsectioned. Dehydration and antigen retrieval were performed before thetissues were incubated with rabbit anti-ANO1 (anoctamin 1) antibody at1:500 dilution, followed by Alexa fluor conjugated secondary antibody,with 488 nm excitation and emission at 515 nm.

Intracellular Calcium and cAMP Measurements

Intracellular calcium levels were measured in isolated mouse smallintestinal cells loaded with Fluo-8 AM dye and incubated for 45 minutes.Fluorescent images were captured with a scanning confocal microscopefitted with argon lasers with an excitation of 488 nm and emission of515 nm wavelengths. Intracellular cAMP measurements were made on celllysates from freshly isolated ileal epithelial cells using a cAMP directimmunoassay (Calbiochem, EMD Millipore, Billerica, Mass., USA)

Western Blot Analysis

Thirty micrograms of protein were resolved by electrophoresis throughSDS-7% polyacrylamide gels. Blots were subsequently reacted with rabbitanti-ANO1 antibody at 1-2 ug/ml, followed by peroxidase-coupledsecondary antibody at a 1:3000 dilution. Immunoreactive bands werevisualized by enhanced chemiluminescence and autoradiography.

Results

The results, as shown in FIGS. 4-9, show that exposure to radiationresults in a dose-dependent increase in Isc and a time-dependentincrease in anion secretion in mouse small intestine. Irradiation alsoincreases intracellular calcium levels but does not increase cAMP levelsin ileal epithelial cells. Also, irradiation increases ANO1 expressionlevels in the brush border membrane of villous epithelial cells. Westernblot analysis showed increased ANO1 protein levels in BBMV of ilealepithelial cells following irradiation, and increased ANO1 protein levelin RBC membranes of mice that received irradiation.

The results show that ANO1 is an important transporter mediating theradiation-induced diarrhea. Also, ANO1 protein levels in RBC membraneincreases with radiation exposure; therefore, ANO1 expression levels inRBC membrane can be used as a surrogate marker for acutegastrointestinal toxicity.

Example 3 The Use of Enzyme-Linked Immunosorbent Assay (ELISA) forDetermining ANO1 Level

In a preferred embodiment, the ANO1 level is determined using theenzyme-linked immunosorbent assay (ELISA), which can be used todetermine the level of ANO1 in both native and denatured conformations.

In one embodiment, procedures for performing ELISA are illustrated asfollows:

A “capture” antibody raised against ANO1 is immobilized onto the surfaceof a polystyrene 96-well microtiter plate. The unbound areas of thewells are blocked with bovine serum albumin or casein to minimizebackground. A test sample with unknown quantity of ANO1 is applied tothe wells, followed by the application of a “detection” antibody forbinding to ANO1, thereby forming a ternary complex (The “detection”antibody should be directed against an epitope different from that forthe “capture” antibody). Next, an enzyme-linked antibody that recognizesthe “detection” antibody is added, and then the enzyme's substrate isadded. The reaction produces a detectable signal, most commonly a colorchange, as a function of time, proportional to the concentration of theANO1 present in the sample.

In one embodiment of the ELISA method, all steps utilize aqueousreagents. Also, at least two different, specific, antibodies against twodifferent epitopes of ANO1 are used. Each step is followed by 2-3 washeswith phosphate buffered saline (PBS) containing a non-ionic detergent toremove any residual proteins or antibodies that are not specificallybound. All steps are performed at room temperature (sample incubationcan be at 37° C.). The ELISA method also requires a plate reader withthe appropriate wavelength of visible light.

Table 1 lists reagents useful for determining ANO1 level in a sampleusing the ELISA method.

TABLE 1 Reagents useful for ELISA Chemicals Sodium phosphate-monobasicSodium phosphate-dibasic Sodium chloride TWEEN ™ 20 TRITON ™ X-100Bovine Serum Albumen Non-fat dry milk Casein TMB peroxidase substrateHydrogen Peroxide (30% Soln) Ethanol (200 proof) Antibodies EB 08600 EB08583 ProSci 5419 Aviva 42506 HRP-anti rabbit HRP-anti goat

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

We claim:
 1. A method for determining radiation dose absorbed by asubject who has been, or is suspected of having been, exposed toionizing radiation, wherein the method comprises: (a) providing abiological sample from a subject who has been, or is suspected of havingbeen, exposed to ionizing radiation; (b) determining expression level ofanoctamin 1 in the subject's biological sample; and (c) determining theradiation dose absorbed by the subject based on the level of expressiondetermined in step (b).
 2. The method according to claim 1, wherein thebiological sample is a blood sample.
 3. The method according to claim 1,wherein the anoctamin 1 expression level in the subject's biologicalsample is determined by Western blot, enzyme-linked immunosorbent assay(ELISA), immunoprecipitation, reverse transcription polymerase chainreaction (RT-PCR), nucleic acid hybridization, or a combination thereof.4. The method according to claim 3, wherein the anoctamin 1 expressionlevel in the subject's biological sample is determined by ELISA.
 5. Themethod according to claim 1, wherein the subject has been, or issuspected of having been, exposed to ionizing radiation at a dose of atleast 1 Gy.
 6. The method according to claim 1, wherein the subject is ahuman.
 7. A method of determining whether a subject has radiationtoxicity, wherein the method comprises: (a) providing a biologicalsample from a subject who has been, or is suspected of having been,exposed to ionizing radiation; (b) determining anoctamin 1 expressionlevel in the subject's biological sample; and (c) comparing theexpression level determined in step (b) to a level of anoctamin 1expression in a normal control; wherein an increased expression ofanoctamin 1 in the subject's biological sample with respect to thecontrol indicates the subject has radiation toxicity.
 8. The methodaccording to claim 7, wherein the biological sample is a blood sample.9. The method according to claim 7, wherein the anoctamin 1 expressionlevel in the subject's biological sample is determined by Western blot,enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, reversetranscription polymerase chain reaction (RT-PCR), nucleic acidhybridization, or a combination thereof.
 10. The method according toclaim 9, wherein the anoctamin 1 expression level in the subject'sbiological sample is determined by ELISA.
 11. The method according toclaim 7, wherein the subject has been, or is suspected of having been,exposed to ionizing radiation at a dose of at least 1 Gy.
 12. The methodaccording to claim 7, wherein the subject is a human.
 13. The methodaccording to claim 7, wherein the subject had received a predetermineddose of ionizing radiation in a radiation therapeutic regime, andwherein the method further comprises providing a toxicity-monitoredradiation therapeutic regime in the subject comprising the steps of: (d)if the level of anoctamin 1 expression determined in (b) is greater thancontrol, then prescribing additional radiation at a dose lower than thepredetermined dose or discontinuing the radiation therapy; and if thelevel of anoctamin 1 expression determined in (b) is no greater than thecontrol, then continuing the radiation therapy at the predetermineddose.
 14. The method according to claim 7, wherein the subject hadreceived a predetermined dose of ionizing radiation in a radiationtherapeutic regime, and wherein the method further comprises providing atoxicity-monitored radiation therapeutic regime in the subjectcomprising the steps of: before the subject receives the predetermineddose of ionizing radiation in the radiation therapeutic regime,determining anoctamin 1 expression level in a biological sample of thesubject; if the level of anoctamin 1 expression determined in (b) isgreater than 105% of the anoctamin 1 expression level of the subjectbefore the subject receives the predetermined dose of ionizingradiation, then prescribing additional radiation at a dose lower thanthe predetermined dose or discontinuing radiation therapy; and if thelevel of anoctamin 1 expression determined in (b) is no greater than105% of the anoctamin 1 expression level of the subject before thesubject receives the predetermined dose of ionizing radiation, thencontinuing the radiation therapy at the predetermined dose.
 15. A methodfor screening for a therapeutic agent that reduces radiation toxicity,wherein the method comprises: (a) providing a population of cellsexposed to ionizing radiation and having an increased expression ofanoctamin 1, and determining a first level of anoctamin 1 expression inthe population of cells exposed to ionizing radiation; (b) contactingthe population of cells with a candidate therapeutic agent for reducingradiation toxicity; (c) after step (b), determining anoctamin 1expression level in the population of cells contacted with the candidatetherapeutic agent; and (d) selecting the candidate agent that reducesthe level of anoctamin 1 expression as the therapeutic agent thatreduces radiation toxicity.
 16. The method according to claim 15,wherein the candidate agent is contacted with a population of cells of asubject who has been exposed to ionizing radiation.